Rous sarcoma virus (RSV), the first tumor virus identified, has been the genesis for seminal discoveries in cancer biology and retrovirology, including oncogenes, reverse transcription, and fundamental aspects of retrovirus assembly. In our laboratory, we utilize RSV as a model system for dissecting mechanisms that regulate nucleocytoplasmic trafficking of proteins and RNAs during the process of retrovirus assembly. Recently we discovered that the RSV Gag protein, which orchestrates virus assembly from the plasma membrane, unexpectedly shuttles through the nucleus. Using a multidisciplinary approach, we demonstrated that RSV Gag selects its viral RNA genome in the nucleus, suggesting a novel paradigm for retroviral RNA packaging. Furthermore, our results suggest that the decision regarding whether the unspliced RSV RNA will be packaged into a virion or used to synthesize structural viral proteins is made in the nucleus. However, host pathways that influence the cytoplasmic destiny of RSV unspliced RNAs remain poorly understood. A further challenge is to decipher how RSV circumvents cellular mechanisms that block export of intron-containing pre-mRNAs, which are normally prevented from leaving the nucleus as part of the quality control machinery that regulates gene expression. Many of the mechanisms that regulate nuclear RNA export are dysregulated in cancer cells, suggesting that learning how RSV subverts nuclear export pathways may have a direct impact on elucidating how gene expression is controlled in normal and transformed cells. The model that newly synthesized mRNAs have their cytoplasmic fates decided in the nucleus is generally accepted, although how pre-mRNA nuclear retention factors prevent the export of unspliced RNAs represents a significant gap in understanding. To address this question, we used RNA affinity capture to identify nuclear proteins that bound to the retroviral 5' untranslated region (UTR). We identified several regulators of splicing and proteins involved in nuclear pre-mRNA surveillance and retention. Based on these preliminary results, our overarching hypothesis is that pre-mRNA nuclear retention factors compete with the RSV Gag protein for newly transcribed viral RNA to decide whether the RNA becomes destined for genome packaging or translation. We will test this hypothesis in two specific aims. First, we will determine whether the nuclear retention factor U2AF65 (U2 snRNP auxiliary factor large subunit) is involved in marking the population of unspliced RSV RNA destined for use in translation. Second, we will determine whether competition between Tpr, a pre-mRNA nuclear retention factor, and Gag for binding the highly structured 5' UTR acts as a switch that regulates the cytoplasmic fate of the unspliced RSV RNA for translation versus packaging. These experimental results will have a significant impact on understanding cellular mechanisms that prevent the nuclear export of unspliced pre-mRNAs and how retroviruses circumvent these quality control measures to express and package their genomes.